Multi-conjugate of SiRNA and preparing method thereof

ABSTRACT

The present invention relates to a multi-conjugate of small interfering RNA (siRNA) and a preparing method of the same, more precisely a multi-conjugate of siRNA prepared by direct binding of double stranded sense/antisense siRNA monomers or indirect covalent bonding mediated by a cross-linking agent or a polymer, and a preparing method of the same. The preparing method of a siRNA multi-conjugate of the present invention is characterized by simple and efficient reaction and thereby the prepared siRNA multi-conjugate of the present invention has high molecular weight multiple times the conventional siRNA, so that it has high negative charge density, suggesting that it has excellent ionic interaction with a cationic gene carrier and high gene delivery efficiency.

TECHNICAL FIELD

The present invention relates to a multi-conjugate of siRNA (smallinterfering RNA) and a preparing method of the same, more precisely amulti-conjugate of siRNA prepared by direct or indirect covalent bondingof double-stranded sense/antisense siRNA monomers mediated by across-linking agent or a polymer.

BACKGROUND ART

SiRNA (small interfering RNA) is a short double-stranded RNA composed of19-22 nucleic acids, which targets mRNA (messenger RNA) of a gene whosenucleotide sequence is identical with its sense strand in order tosuppress expression of the gene by decomposing the target gene(Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K.,and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cells. Nature 411, 494-8).

SiRNA is capable of inhibiting gone expression even with 10 times lessamount than the required amount of conventional antisenseoligonucleotide, suggesting that it has excellent gene selectivityindicating that it is highly capable of inhibiting a target gene alone.However, siRNA is so unstable in vivo that it is easily decomposedwithin a short period of time and it is anionic which makes cellmembrane transmission difficult, resulting in low intracellular deliveryefficiency.

To increase siRNA delivery efficiency, a nano-sized ion-complex isgenerally used which is prepared by ionic bonding of siRNA and diversefunctional cationic polymers, lipids or cationic peptides. However, theconventional siRNA has the molecular weight of about 15,000 and has avery stiff structure of double strand. So, it is very difficult toprepare a stable siRNA/cationic gene carrier complex (Gary, D. J., Puri,N., and Won, Y. Y. (2007) Polymer-based siRNA delivery: perspectives onthe fundamental and phenomenological distinctions from polymer-based DNAdelivery. J Control Release 121, 64-73).

Therefore, studies have been actively undergoing to prepare a stablesiRNA complex with a gene carrier. It has been attempted to increasecharge density of each element of an siRNA or a cationic carrier toincrease stability of an ionic complex. As an example, it has beenattempted to induce strong ionic interaction by increasing molecularweight of cationic polymer or lipid or by introducing a strong cationicgroup into a cationic carrier. However, even if the said method canincrease gene delivery efficiency, it also increases non-specificcytotoxicity owing to the strong cations of a gene carrier, making theclinical application difficult. So, a new approach has recently beenmade to modify siRNA itself to produce a stable complex with theconventional gene carrier.

According to recent reports, 4-8 additional nucleotides (deoxythymine,deoxyadenine) are added to sense strand in order to increase themolecular weight of siRNA, resulting in complementary base pairing insiRNA complex (Bolcato-Bellemin, A. L., Bonnet, M. E., Creusat, G.,Erbacher, P., and Behr, J. P. (2007) sticky overhangs enhancesiRNA-mediated gene silencing. Proc Natl Acad Sci USA 104, 16050-5).However, at this time, complementary bindings of 4-8 nucleotides are sounstable that it cannot be confirmed by electrophoresis.

Thus, the present inventors continued study to increase stability anddelivery efficiency of siRNA. As a result, the present inventorscompleted this invention by confirming that a multi-conjugate of siRNAprepared by direct or indirect covalent bonding of double-strandedsense/antisense siRNA monomers mediated by a cross-linking agent or apolymer has excellent gene delivery efficiency owing to strong ionicbond with a cationic gene carrier and does not induce severe immuneresponse, compared with the conventional siRNA, so that it can beeffectively used for gene therapy.

DISCLOSURE Technical Problem

It is an object of the present invention to provide a multi-conjugate ofsiRNA having higher gene inhibition efficiency than the conventionalsiRNA.

It is another object of the present invention to provide a simple buthighly efficient preparing method of a multi-conjugate of siRNA that iscapable of binding stably with a gene carrier owing to the highnegative-charge density.

Technical Solution

To achieve the above objects, the present invention provides amulti-conjugate of siRNA having the following structure of [StructuralFormula I] or [Structure Formula II] prepared by direct or indirectcovalent bonding of double-stranded sense/antisense siRNA monomersmediated by a cross-linking agent or a polymer:(-X-A-)_(n)  [Structural Formula I]

(Wherein,

X is double-stranded siRNA monomer;

A is either presented or not presented, a cross-linking agent or apolymer; and

n is the number of double-stranded siRNA monomer),x-A-(X-A-)_(n)-X′  [Structural Formula II]

(Wherein,

X is double-stranded siRNA monomer;

x or x′ is single-stranded siRNA monomer

A is either presented or not presented, a cross-linking agent or apolymer; and

n is the number of double-stranded siRNA monomer).

The present invention also provides a preparing method of amulti-conjugate of siRNA having the structure of [Structural Formula I]or [Structural Formula II] containing the step of direct or indirectcovalent bonding of double-stranded sense/antisense siRNA monomersmediated by a cross-linking agent or a polymer.

The present invention further provides an ionic complex formed by ionicinteraction between the said siRNA multi-conjugate and a cationic genecarrier selected from the group consisting of cationic peptides,cationic lipids and cationic polymers.

The present invention also provides a method for treating cancer orangiogenesis related disease containing the step of administering thesaid ionic complex to a subject.

In addition, the present invention provides a use of the said ioniccomplex for the production of an anticancer agent or a therapeutic agentfor angiogenesis related disease.

Hereinafter, the present invention is described in detail.

The present invention provides a multi-conjugate of siRNA having thefollowing structure of [Structural Formula I] or [Structure Formula II]prepared by direct or indirect covalent bonding of double-strandedsense/antisense siRNA monomers mediated by a cross-linking agent or apolymer:(-X-A-)_(n)  [Structural Formula I]

(Wherein,

X is double-stranded siRNA monomer;

A is either presented or not presented, a cross linking agent or apolymer; and

n is the number of double-stranded siRNA monomer),x-A-(X-A-)_(n)-x′  [Structural Formula II]

Wherein,

X is double-stranded siRNA monomer;

x or x′ is single-stranded siRNA monomer

A is either presented or not presented, a cross-linking agent or apolymer; and

n is the number of double-stranded siRNA monomer).

In this invention, the said double-stranded sense/antisense siRNAmonomer preferably has 15-50 nucleotides and more preferably has 15-23nucleotides, but not always limited thereto.

In this invention, the number of the said double-strandedsense/antisense siRNA monomer is preferably 1-50 and more preferably1-100, but not always limited thereto.

The multi-conjugate of siRNA of the present invention is preferablyprepared by the following two methods, but not always limited thereto:

1) The first method: single-stranded sense siRNA and antisense siRNAeach having a functional group at terminal are reacted respectively inthe presence of a cross-linking agent ox a polymer to producesingle-stranded sense and antisense siRNA dimers. Each dimer iscomplementarity annealed in aqueous solution (see FIGS. 1B and 1D).

Particularly, sense strand siRNA and antisense strand siRNA each havingsulfhydryl group substituted at terminal are reacted to produce dimersby cleavable or non-cleavable covalent bond using DTME(Dithio-bis-maleimidoethane) or BM(PEG)₂ (1,8-bis(maieimido)diethyieneglycol), the cross-linking agent reactable with sulfhydryl group. Then,equal amount of each dimer is loaded in PBS to induce complementarybinding via hydrogen bond, resulting in the preparation of a siRNAmulti-conjugate.

2) The second method: double-stranded siRNA (prepared by complementaryhydrogen bond) having a functional group substituted at terminalproceeds to covalent bond mediated by a cross-linking agent or apolymer, resulting in the preparation of a siRNA multi-conjugate (seeFIGS. 1A and 1C).

Particularly, single-stranded siRNA introduced with sulfhydryl group atterminal proceeds to complementary hydrogen bond, and the resultantproduct proceeds to covalent bond based on oxidation in the presence ofa cross-linking agent or DMSO, resulting in the preparation of a siRNAmulti-conjugate.

In the production of the said multi-conjugate of siRNA, the molecularweight of siRNA oligo-strand is preferably selected in the range from10,000-50,000, but not always limited thereto. The siRNA for the presentinvention is not limited and any one that is being used for treatment orstudy can be accepted, for example any siRNA that is being used for genetherapy or study or is expected to be used in near future is selected,which is exemplified by c-myc, c-myb, c-fos, c-jun, bcl-2 or VEGF,VEGF-B, VEGF-C, VEGF-D, PIGF, etc.

Hydroxy group (—OH) at terminal of the said siRNA can be substitutedwith a functional group such as sulfhydryl group (—SH), carboxyl group(—COOH) or amine group (—NH₂),

The substitution can be performed at 3′ and or 5′ end, and is preferablyoccurred at 3′ ends of both sense and antisense are substituted withsuch functional group, but not always limited thereto.

The polymer herein can be a non-ionic hydrophilic polymer such aspolyethyleneglycol (PEG), polyvinylpyrrolidone and polyoxazolin, or ahydrophobic polymer such as PLGA and PLA, etc.

The cross-linking agent herein has the molecular weight of 100-10000,which is exemplified by DTME (Dithio-bis-maleimidoethane), BM(PEG)₂(1,8-Bis-maleimidodiethyleneglycol), maleimide, NHS(N-hydroxysuccinimide), vinylsulfone, iodoacetyl, nitrophenyl azide,isocyanate, pyridyldisulfide, hydrazide or hydroxyphenyl azide, but notalways limited thereto.

Either the cross-linking agent having external stimulus-mediatedcleavable bonds or the cross-linking agent having non-cleavable bondscan be used herein, for example, the cross-linking agent of the presentinvention can have non-cleavable bond such as amide bond and urethanebond, and cleavable bond such as acid cleavable bond (covalent bond ofester, hydrazone, acethal, etc) and reductant cleavable bond (disulfidebond), etc. And additionally, any cross-linking agent available for drugmodification can be used without limitation.

The said multi-conjugate of siRNA can include cell selective ligand atthe end.

The ligand can be selected from the group consisting of cell specificantibody, cell selective peptide, cell growth factor, folic acid,galactose, mannose, RGD, and transferrin. Such ligand can be introducedat the end of the multi-conjugate by disulfide bond, amide bond, orester bond, etc.

The multi-conjugate of siRNA of the present invention can form an ioniccomplex by ionic interaction with a cationic gene carrier (cationiclipid, cationic polymer, cationic peptide, etc).

The cationic peptide is KALA (cationic fusogenic peptide), polylysine,polyglutamic acid or protamine. The KALA preferably has the peptidesequence of WEAKLAKALAKALAKHLAKALAKALAACEA (SEQ. ID. NO: 1), but notalways limited thereto.

The cationic lipid is dioleyl phosphatidylethanolamine or cholesteroldioleyl phosphatidylcholine.

The cationic polymer is polyethyleneimine, polyamine or polyvinylamine.

To investigate target gene inhibition efficiency of the multi-conjugateof siRNA of the present invention, the inventors mixed siRNA with thegene carrier Linear PEI to prepare an ionic complex, which was thentreated to cancer cells expressing GFP stably. Then, GFP level wasmeasured using fluorophotometer. As a result, the multi-conjugate ofsiRNA of the present invention demonstrated higher gene deliveryefficiency using a cationic gene carrier than conventional siRNA,suggesting that the multi-conjugate of siRNA of the present inventionhad excellent target gene inhibition efficiency (see FIG. 4).

To investigate binding force with a cationic gene carrier and stabilityof the siRNA multi-conjugate of the present invention, the inventorsproduced an ionic complex by mixing the gene carrier Linear PEI andsiRNA, followed by observation of shape and size using Atomic ForceMicroscopy (AFM). As a result, compared with conventional siRNA, thesiRNA multi-conjugate or the present invention demonstrated excellentbinding force with a cationic polymer and had smaller size which favoredproduction of even and small nano particles (see FIG. 5).

To investigate the amount of a cationic polymer conjugated to the siRNAmulti-conjugate of the present invention, gel retardation assay wasperformed. As a result, compared with conventional siRNA, the siRNAmulti-conjugate of the present invention demonstrated higher chargedensity, which favored production of an ionic complex by binding acationic polymer even at a low concentration (see FIG. 6).

The present inventors also investigated gene inhibition efficiency ofthe siRNA multi-conjugate of the present invention. To do so, theinventors produced an ionic complex by mixing the gene carrier LinearPEI with siRNA, which was treated to cancer cells. Then, VEGF level wasmeasured by ELISA. As a result, compared with conventional siRNA, thesiRNA multi-conjugate of the present invention formed an ionic complexwith a cationic polymer more stably and evenly, suggesting that themulti-conjugate of siRNA of the present invention had excellent genedelivery efficiency and excellent selective inhibition of a target gene(see FIG. 7).

The present inventors also investigated gene inhibition efficiency ofthe siRNA multi-conjugate of the present invention according to themolecular weight. First, siRNA was separated over the size by gelseparation method. Each separated siRNA was mixed with Linear PEI toproduce an ionic complex. Then, the complex was treated to cancer cells,followed by quantification of VEGF by ELISA. As a result, as themolecular weight of the siRNA multi-conjugate of the present inventionwas increased, charge density was increased, and accordingly theefficiency of gene delivery using a cationic polymer was also increased(see FIG. 8).

The present inventors also investigated immune response inducing abilityof the siRNA multi-conjugate of the present invention. The presentinventors produced an ionic complex by mixing the cationic gene carrierLinear PEI, jet PEI or DOTAP with siRNA, which was treated to humanPBMC. Then, interferon-α (INF-α) was quantified by ELISA. As a result,there was no significant induction of INF-α by the siRNA multi-conjugateof the present invention (see FIG. 9A).

To investigate immune response inducing capacity of the siRNAmulti-conjugate of the present invention, the inventors produced anionic complex by mixing the cationic gene carrier Linear PEI with siRNA,which was intravenously injected to ICR mouse at 7 weeks. Blood wastaken from the heart of the moose, followed by quantification of bloodsiRNA by ELISA. As a result, in this animal model, there was nosignificant induction of INF-α by the siRNA multi-conjugate of thepresent invention, compared with conventional siRNA. (see FIG. 9B).

Therefore, the siRNA multi-conjugate of the present invention wasconfirmed to have higher molecular weight, higher negative chargedensity, higher stability, and higher ionic strength with a cationicgene carrier than conventional siRNA, resulting in high gene deliveryefficiency. The siRNA multi-conjugate of the present invention can besimply produced by using the therapeutic gene siRNA alone, which favorsbiocompatibility. Moreover, even as multi-conjugate form, the siRNA ofthe present invention maintains gene inhibition activity but hasincreased gene delivery efficiency, suggesting that it has higherintracellular introduction efficiency and thus higher gene inhibitioneffect than conventional siRNA.

VEGF binds to VEGF receptor existing on vascular endothelial cellsurface and is then functioning to accelerate growth and migration ofendothelial cell and angiogenesis. In particular, tumor growth andmetastasis are closely related to angiogenesis. So, inhibition ofangiogenesis can be a new approach to treat cancer by inhibiting thegrowth of tumor. The siRNA multi-conjugate of the present invention caninhibit VEGF significantly, so that it can be effectively used fortreating cancer.

The present invention also provides a preparing method of amulti-conjugate of siRNA having the structure of [Structure Formula I]or [Structure Formula II] containing the step of direct or indirectcovalent bonding of double-stranded sense/antisense siRNA monomersmediated by a cross-linking agent or a polymer.

In a preferred embodiment of the present invention, the preparing methodof the multi-conjugate of siRNA is composed of the following steps:

1) preparing yXy by complementary hydrogen bonding of single-strandedsense/antisense siRNA monomers (yx+x′y) having substitutions with samefunctional groups at the ends; and

2) inducing covalent bond of the yXy mediated by a cross-linking agentor a polymer (Herein, X is double-stranded siRNA monomer, x and x′ aresingle-stranded sense/antisense siRNA monomers, and y is the functionalgroup introduced in the end.).

In another preferred embodiment of the present invention, the preparingmethod of the multi-conjugate of siRNA is composed of the followingsteps:

1) preparing dimers of xyyz and x′yyx′ by covalent bonding ofsingle-stranded sense/antisense siRNA monomers (yx and x′y) havingsubstitutions with same functional groups at the ends; and

2) inducing complementary hydrogen bond between the dimers xyyx andx′yyx′ (Herein, X is double-stranded siRNA monomer, x and x′ aresingle-stranded sense/antisense siRNA monomers, and y is the functionalgroup introduced in the end.).

In another preferred embodiment of the present invention, the preparingmethod of the multi-conjugate of siRNA is composed of the followingsteps:

1) preparing yXz by complementary hydrogen bonding of single-strandedsense/antisense siRNA monomers (yx+x′z) having substitutions withdifferent functional groups at the ends; and

2) inducing covalent bond of the yXz mediated by a cross-linking agentor a polymer (Herein, X is double-stranded siRNA monomer, x and x′ aresingle-stranded sense/antisense siRNA monomers, and y and z are thefunctional groups introduced in the ends.).

In another preferred embodiment of the present invention, the preparingmethod of the multi-conjugate of siRNA is composed of the followingsteps:

1) preparing dimers of xyzx and x′yzx′ by covalent bonding ofsingle-stranded sense/antisense siRNA monomers (yx and xz, x′y and x′z)having substitutions with different functional groups at the ends; and

2) inducing complementary hydrogen bond between the dimers xysx andx′yzx′ (Herein, X is double-stranded siRNA monomer, x and x′ aresingle-stranded sense/antisense siRNA monomers, and y and z are thefunctional groups introduced in the ends.).

In the preparing method of a multi-conjugate of siRNA of the presentinvention, siRNA oligo strand is preferably selected from those havingthe molecular weight of 10,000-50,000. The siRNA herein is not limitedand any siRNA that is used for treatment or study can be accepted, forexample any siRNA that has been used for gene therapy or study or isexpected to be used for that purpose can be accepted, which isexemplified by c-myc, c-myb, c-fos, c-jun, bcl-2 or VEGF, VEGF-B,VEGF-C, VEGF-D, PIGF, etc.

In the preparing method of a multi-conjugate of siRNA of the presentinvention, the covalent bond is preferably selected from the groupconsisting of non-cleavable bonds (amide bond and urethane bond), acidcleavable bonds (ester bond, hydrazone bond and acethal bond), reductantcleavable bond (disulfide bond), bio-cleavable bonds and enzymecleavable bonds, but not always limited thereto.

In the preparing method of a multi-conjugate of siRNA of the presentinvention, hydroxyl group (—OH) at the end of the single-strandedsense/antisense siRNA monomer is preferably substituted with one of thefunctional groups selected from the group consisting of sulfhydryl group—SH), carboxyl group (—COOH) and amine group (—NH₂), but not alwayslimited thereto.

The substitution is preferably performed at 3′ end or 5′ end, and it ispreferred that sense and antisense are substituted with functionalgroups at 3′ ends, but not always limited thereto.

In the preparing method of a multi-conjugate of siRNA of the presentinvention, the polymer used as a mediator for covalent bond ispreferably one or more non-ionic hydrophilic polymers selected from thegroup consisting of PEG, Pluronic, polyvinylpyrrolidone andpolyoxazolin; or one or more biocleavable polyester polymers selectedfrom the group consisting of poly-L-lactic acid, poly-glycolic acid,poly-D-lactic-co-glycolic acid, poly-L-lactic-co-glycolic acid,poly-D,L-lactic-co-glycolic acid, polycaprolactone, polyvalerolactone,polyhydroxybutyrate and polyhydroxyvalerate, but not always limitedthereto.

In the preparing method of a multi-conjugate of siRNA of the presentinvention, the cross-linking agent is one or more compounds having themolecular weight of 100-10,000, which is preferably selected from thegroup consisting of DTME (Dithio-bis-maleimidoethane), BM(PEG)₂(1,8-Bis-maleimidodiethyieneglycol), maleimide, NHS(N-hydroxysuccinimide), vinylsulfone, iodoacetyl, nitrophenyl azide,isocyanate, pyridyldisulfide, hydrazide and hydroxyphenyl azide, but notalways limits thereto.

In the preparing method of a multi-conjugate of siRNA of the presentinvention, the cell selective ligand is preferably added to the end ofthe said siRNA multi-conjugate, but not always limited thereto.

The said ligand is one or more compounds preferably selected from thegroup consisting of cell specific antibody, cell selective peptide, cellgrowth factor, folic acid, galactose, mannose, RGD and transferrin, butnot always limited thereto.

In the preparing method of a multi-conjugate of siRNA of the presentinvention, the step of activating the functional group of the said siRNAcan be additionally included, but not always limited thereto.

The material used for the activation of the functional group of the saidsiRNA is one or more compounds preferably selected from the groupconsisting of 1-ethyl-3,3-dimethylaminopropyl carbodiimide, imidazole,N-hydroxysuccinimide, dichlorohexylcarbodiimide, N-β-Maleimidopropionicacid, N-β-maleimidopropyl succimimide ester and N-Succinimidyl3-(2-pyridyldithio)propionate, but not always limited thereto.

In the preparing method of a multi-conjugate of siRNA of the presentinvention, the preparation reaction is not limited, bat generallyperformed at room temperature for about 24-48 hours. Ratio of eachreactant added to the reaction is not limited and the length, of siRNAmulti-conjugate can be regulated by adjusting molar ratio (%) of across-linking agent to siRNA.

The aqueous solution used, for complementary binding in this inventionis PBS, Tris buffer or HEPES buffer, and more preferably the bufferhaving salt concentration of at least 100 mM.

The present invention also provides an ionic complex facilitatingcellular transduction, which is formed by ionic interaction between thesaid siRNA multi-conjugate and a cationic gene carrier selected from thegroup consisting of cationic peptides, cationic lipids and cationicpolymers.

An ionic complex can be prepared by ionic interaction between the siRNAmulti-conjugate of the present invention and a cationic gene carrier.The said ionic complex is a small complex formed by interaction betweenan anionic gene and a polymer having counter ions (ex, cationicmaterial), which is preferably 100-200 nm in size, but not alwayslimited thereto.

In this invention, the cationic peptide is preferably selected from thegroup consisting of KALA (cationic fusogenic peptide), polylysine,polyglutamic acid and protamine, but not always limited thereto.

In this invention, the cationic lipid is preferably selected from thegroup consisting of dioleyl phosphatidylethanolamine and cholesteroldioleyl phosphatidyl choline, but not always limited thereto.

In this invention, the cationic polymer is preferably selected from thegroup consisting of polyethylenimine, polyamines and polyvinylamine, butnot always limited thereto.

In the preparation of the ionic complex, the siRNA multi-conjugate ofthe present invention is diluted in PBS, to which a cationic substanceis added. Then, the mixture stands at room temperature, resulting in thepreparation of an ionic complex in aqueous solution. At this time, thecontent of the cationic substance is regulated by adjusting the ratio ofpositive charge of the cationic substance to negative charge of thesiRNA to 1:1 (+/−=1/1)˜100:1.

The present invention also provides a method for treating cancer orangiogenesis related disease containing the step of administering thesaid ionic complex to a subject.

In addition, the present invention provides a use of the said ioniccomplex for the production of an anticancer agent or a therapeutic agentfor angiogenesis related disease.

The term “subject” herein indicates human or animals including monkey,dog, goat, pig and mouse that have cancer or angiogenesis relateddisease that can be improved by the administration of the ionic complexof the present invention.

The term “administration” herein indicates providing a required amountof the ionic complex of the present invention to a subject through aproper method.

The term “treatment” herein indicates all behavior performed to improveor relieving symptoms of cancer or angiogenesis related disease byadministering the ionic complex of the present invention.

The cancer herein is preferably selected from, the group consisting ofbreast cancer, colorectal cancer, rectal cancer, non-small cell lungcancer, non-Hodgkin's lymphoma (NHL), renal cell cancer, prostatecancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi'ssarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovariancancer, mesothelioma and multiple myeloma, but not always limitedthereto.

The angiogenesis related disease herein is selected from the groupconsisting of hemangioma, angiofibroma, arthritis, diabetic retinosis,retrolental fibroplasia, neovascular glaucoma, neovascular cornealdisease, macular degeneration, macular dystrophy, pterygium, retinaldegeneration, retrolental fibroplasia, trachoma, psoriasis,capillarectasia, granuloma pyogenicum, dermatitis seborrheica and acne,but not always limited thereto.

The siRNA multi-conjugate of the present invention is prepared by siRNAalone, so that it has excellent biocompatibility and excellent stabilityand improved binding capacity with various gene carriers withoutreducing gene inhibition activity of siRNA, suggesting that the siRNAmufti-conjugate of the present invention has higher gene inhibitionactivity than conventional siRNA.

In this invention, an ionic complex prepared by mixing a siRNAmulti-conjugate and the representative gene carrier Linear PEI wastreated to cancer cells. As a result, significant VEGF inhibition effectwas demonstrated, compared with conventional siRNA. That is, the siRNAmulti-conjugate of the present invention forms a very stable and regularionic complex with a cationic polymer and therefore the conjugatedemonstrates excellent gene delivery effect and target gene inhibitioneffect.

Therefore, the ionic complex of the present invention can be used fordiverse gene therapy, because the siRNA multi-conjugate can includediverse siRNA, in particular for gene therapy for cancer or angiogenesisrelated disease.

Advantageous Effect

The preparing method of a multi-conjugate of siRNA of the presentinvention is characterized by simple reaction process and highefficiency. Therefore, the siRNA multi-conjugate prepared by the abovemethod has the molecular weight of multiple times the conventionalsiRNA, and thus has higher negative charge density, indicating that thesiRNA multi-conjugate of the present invention has significantly highgene delivery efficiency resulted from excellent ionic interaction witha cationic gene carrier.

DESCRIPTION OF DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a diagram illustrating the preparing method of amulti-conjugate of siRNA. (A) Double-stranded siRNA having functionalgroups introduced in both ends is prepared by complementary base-pairingvia hydrogen bonding of single-stranded sense siRNA and single-strandedantisense siRNA having same functional group substituted at one end.Then, a multi-conjugate of siRNA is prepared by covalent bonding using across-linking agent or a polymer, (B) Dinner form of sense strand andantisense strand siRNA is first prepared by covalent bondingsingle-stranded sense siRNA and single-stranded antisense siRNA havingsame functional group substituted at one end using a cross-linking agentor a polymer. Then, a multi-conjugate of siRNA is prepared bycomplementary hydrogen bonding of each oligonucleotide. (C)Double-stranded siRNA having different functional groups introduced inboth ends is prepared by complementary base-pairing via hydrogen bondingof single-stranded sense siRNA and single-stranded antisense siRNAhaving different functional group at each end. Then, a multi-conjugateof siRNA is prepared by direct covalent bonding or covalent bondingmediated by a cross-linking agent. (D) Dimer form of sense strand andantisense strand siRNA is first prepared by direct covalent bonding ofsingle-stranded sense siRNA and single-stranded antisense siRNA havingdifferent functional group at each end or covalent bonding mediated by across-linking agent. Then, a multi-conjugate of siRNA is prepared bycomplementary base-pairing via hydrogen bonding of each oligonucleotide,

FIG. 2 illustrates the result of electrophoresis of the multi-conjugateof siRNA. Double-stranded siRNA is prepared by hydrogen bonding betweensense strand siRNA and antisense strand siRNA both having thesubstitution with sulfhydryl group at 3′ end. Then, (A) amulti-conjugate of siRNA is prepared by using the cross-linking agentDTME or (B) a multi-conjugate of siRNA is prepared by using thecross-linking agent BM(PEG)₂ (by the method of FIG. 1A).

FIG. 3(A) illustrates the result of electrophoresis of themulti-conjugate of siRNA prepared by using double-stranded siRNA havingthe substitution with sulfhydryl group at 3′ ends of both sense andantisense siRNA. (B) illustrates the result of electrophoresis of themulti-conjugate of siRNA prepared by complementary binding of dimersproduced from sense and antisense siRNA having substitution withsulfhydryl group at 3′ end in the presence of a cross-linking agent. (C)and (D) illustrate observation of dimer by electrophoresis afterpreparing the dimer by using sense and antisense siRNA of the dimer inthe presence of a cross-linking agent that is capable of linking sensesiRNA and antisense siRNA having substitution with sulfhydryl group at3′ end by cleavable covalent bond which is disulfide bond (C) or bynon-cleavable covalent bond (D).

FIG. 4 illustrates that a complex is prepared with the siRNAmulti-conjugate to GFP of FIG. 1A and FIG. 1B and the conventional siRNAusing the cationic gene carrier linear PEI at NP ratio 20. Then, thecomplex is treated to the cancer cell line MDA-MB-435 expressing GFPstably, followed by quantification of GFP inhibition.

FIG. 5 illustrates that an ionic complex is prepared from the siRNAmulti-conjugate (prepared by the method of FIG. 1B) and the conventionalsiRNA (Naked) using the cationic gene carrier linear PEI at NP ratio 2and 10, and then the size and shape are observed by atomic forcemicroscopy (AFM).

FIG. 6 illustrates that ion complexes are prepared by using the siRNAmulti-conjugate (prepared by the method of FIG. 1B) and the conventionalsiRNA (leaked) using the cationic gene carrier linear PEI over differentNP ratios, followed by electrophoresis to observe the ionic complexformation.

FIG. 7 illustrates that a siRNA multi-conjugate linked by cleavabledisulfide bond or non-cleavable covalent bond is prepared by the methodof FIG. 1B using siRNA inhibiting VEGF (Vascular endothelial growthfactor), followed by comparison of gene inhibition efficiency with theconventional siRNA (Naked). Linear PEI is used as a gene carrier. Geneinhibition efficiency is measured over the concentration of siRNA (A)and over the NP ratio (B) by ELISA, by which VEGF level is quantified.(C) illustrates the result of quantification of mRNA by RT-PCB toinvestigate gene inhibition efficiency.

FIG. 8(A) illustrates that a siRNA multi-conjugate is prepared by themethod of FIG. 1B and each siRNA is separated, followed byelectrophoresis. (B) illustrates that gene inhibition efficiency of eachseparated multi-conjugate is compared by quantifying VEGF by ELISA.

FIG. 9 illustrates the IFN-alpha induction by the siRNA multi-conjugateprepared by the method of FIG. 1B, (A) illustrates that an ionic complexis prepared by using the conventional siRNA or the siRNA multi-conjugatelinked by cleavable or non-cleavable covalent bond and a cationic genecarrier (Linear PEI, Jet-PEI or DOTAP). The ionic complex is introducedinto PBMC (Peripheral Blood Mononuclear Cell) extracted from humanblood, followed by quantification of IFN-alpha released from the cell byELISA. (B) illustrates that an ionic complex is prepared by using theconventional siRNA or the siRNA multi-conjugate linked by cleavable ornon-cleavable covalent bond and the cationic gene carrier Linear PEI,which is intravenously injected into ICR mouse, followed byquantification of IFN-alpha released from blood by ELISA.

MODE FOR INVENTION

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will, be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1: Preparation of Double-Stranded siRNA Via Hydrogen Bond ofSense Strand siRNA and Antisense Strand siRNA Having Substitution withSame Functional Group at their Ends and Preparation of Multi-Conjugateof siRNA Using Cross-Linking Agent

100 nmol of sense or antisense strand siRNA having substitution withsulfhydryl group at 3′ end was dissolved in 260 μl of 1×PBS, which stoodat 37° C. for 1 hour, resulting in double-stranded siRNA. To reducesulfhydryl group at both ends of the prepared double-stranded siRNA, 22μl of 25×PBS, 260 μl of 2M DTT (dithiothreitol) solution and 4 μl of 5NNaOH solution (to adjust pH) were added thereto, followed by reactionfor 12 hours. Upon completion of the reaction, remaining DTT waseliminated by dialysis and the solution was concentrated to 1 nmol/μl.25×PBS was added to adjust the final concentration to 5×PBS. Thecross-linking agent DTME or BM(PEG)₂ was added at the concentration ofhalf the concentration of thiol group, followed by reaction at roomtemperature for 24 hours. Upon completion of the reaction, remainingforeign materials such as cross-linking agent, etc, were eliminated bydialysis and the solution was concentrated to make the finalconcentration to 1˜2 μg/μl to prepare a siRNA multi-conjugate (see FIG.1A). The prepared siRNA was confirmed by electrophoresis. (see FIG. 2)

A multi-conjugate was prepared by direct covalent bonding ofdouble-stranded siRNA mediated by oxidation without using across-linking agent. Double-stranded siRNA having substitution withthiol group at 3′ ends of sense and antisense strands was treated withDTT by the same manner as described above, followed by dialysis andconcentration to make the final concentration of the solution to 1nmol/μl. DMSO and diamide were added to the above solution to oxidizesulfhydryl, resulting in the formation of disulfide bond. The prepareddouble-stranded siRNA multi-conjugate was confirmed by electrophoresis(see FIG. 3A).

Example 2: Preparation of Dimer of Each Sense Strand siRNA and AntisenseStrand siRNA Having the Substitution with Same Functional Group at theEnd Using Cross-Linking Agent and Preparation of siRNA Multi-ConjugateVia Hydrogen Bond

100 nmol of sense or antisense strand siRNA having the substitution withsulfhydryl group at 3′ end was dissolved in 260 μl of DEPC (Diethylpyrocarbonate) treated deionized water, to which 22 μl of 25×PBS wasadded. 260 μl of 2M DTT (dithiothreitol) was added thereto and then 4 μlof 5N NaOH was added to adjust pH, followed by reaction for 12 hours.Upon completion of the reaction, remaining DTT was eliminated bydialysis and the solution was concentrated. As a result, sense orantisense strand siRNA having the final concentration of 1 nmol/μl wasprepared. 25×PBS was added to adjust the final concentration to 5×PBS.The cross-linking agent DTME or BM(PEG)₂ was added thereto at theconcentration of half the concentration of thiol group, followed byreaction at room temperature for 24 hours. Upon completion of thereaction, foreign materials such as cross-linking agent, etc, wereeliminated by dialysis, and the solution was concentrated to prepare thedimer form of sense or antisense siRNA having the final concentration of1˜2 μg/μl (see FIG. 1B). The dimer prepared by cleavable disulfide bond(see FIG. 3C) or non-cleavable covalent bond (see FIG. 3D) wereconfirmed by electrophoresis. Equal amount of sense and antisense dimersstood in PBS at 37° C. for 1 hour to induce hydrogen bond. As a result,a siRNA multi-conjugate was prepared and confirmed by electrophoresis(see FIG. 3B).

Example 3: Preparation of Dongle-Stranded siRNA by Hydrogen Bonding ofSense Strand siRNA and Antisense Strand siRNA Having DifferentFunctional Groups at their Ends and Preparation of siRNA Conjugate UsingCross-Linking Agent

Sense strand and antisense strand siRNA having respectively amine groupand sulfhydryl group at 3′ end were prepared. 100 nmol of each sense andantisense siRNA was dissolved in 260 μl of PBS, which stood at 3° C. for1 hour, resulting in the preparation of double-stranded siRNA, DTT wastreated thereto in order to prepare single-stranded siRNA havingsulfhydryl group substituted at the end, followed by dialysis andconcentration to make the final concentration of 1 nmol/μl. Thecross-linking agent sulfo-SMCC(sulfosuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxylate) wasadded to the prepared double stranded siRNA, followed by reaction for 24hours to prepare a multi-conjugate of siRNA. Upon completion of thereaction, remaining foreign materials such as cross-linking agent, etc,were eliminated by dialysis and the solution was concentrated to makethe final concentration to 1˜2 μl (see FIG. 1C).

Example 4: Preparation of Dimer of Each Sense Strand siRNA and AntisenseStrand siRNA Having the Substitution with Different Functional Groups attheir Ends Using Cross-Linking Agent and Preparation of siRNAMulti-Conjugate Via Hydrogen Bond

Sense and antisense siRNA each having amine group and sulfhydryl groupat 3′ end were linked to make double-stranded siRNA using thecross-linking agent sulfo-SMCC. Single-stranded siRNA having thesubstitution with sulfhydryl group at the end was treated with DTT,followed by dialysis and concentration until the final concentrationreached 1 nmol/μl. Single-stranded siRNA having the substitution withamine group at the end was dissolved in DEPC treated distilled water atthe concentration of 1 nmol/μl. Each solution containing sense andantisense having respectively amine group and sulfhydryl group wastreated with sulfo-SMCC to prepare sense or antisense dimer. Theprepared sense or antisense dimer was mixed in PBS, which stood at 37°C. for 1 hour, resulting in the preparation of a double-stranded siRNAmulti-conjugate (see FIG. 1D).

Experimental Example 1: Measurement of GFP Expression

An ionic complex was prepared from the siRNA multi-conjugate (preparedby the method of FIG. 1A) linked by cleavable disulfide bond ornon-cleavable covalent bond using the siRNA inhibiting GFP gene, theconventional siRNA, a cross linking agent and linear PEI(polyethyleneimine). The prepared ionic complex was treated to thecancer cell line MDA-MB-435 expressing GFP stably for 5 hours. 48 hourslater, GFP expression was quantified with a fluorophotometer.

As a result, the siRNA multi-conjugate of the present inventiondemonstrated excellent gene delivery efficiency using a cationic genecarrier and excellent target gene inhibition activity, compared with theconventional siRNA (see FIG. 4).

Experimental Example 2: Measurement of Binding Strength with CationicGene Carrier and Stability

To confirm whether the siRNA mufti-conjugate (prepared by the method ofFIG. 1B) had excellent binding strength with a cationic gene carrier andcould form a stable ionic complex, compared with the conventional siRNA,the present inventors mixed the representative gene carrier linear PEIand each siRNA to produce each ionic complex. And then, shape and sizeof each complex were observed by AFM.

As a result, the siRNA multi-conjugate of the present inventiondemonstrated excellent binding strength with a cationic polymer and wascapable or forming small but even nano particles, compared with theconventional siRNA (see FIG. 5).

To investigate the amount of the cationic polymer binding to each siRNA,gel retardation assay was performed.

As a result, the siRNA multi-conjugate of the present invention hadhigher charge density than the conventional siRNA, suggesting that thesiRNA multi-conjugate of the present invention can form an ionic complexby binding with a cationic polymer even at a low concentration (see FIG.6).

Experimental Example 3: Investigation of Gene Inhibition EfficiencyUsing VEGF

An ionic complex was prepared from the siRNA multi-conjugate (preparedby the method of FIG. 1B) linked by cleavable disulfide bond ornon-cleavable covalent bond using the siRNA inhibiting VEGF gene, theconventional siRNA, and linear PEI (polyethyleneimine). The preparedionic complex was treated to PEI cancer cells for 5 hours. Then VEGFsecreted for 21 hours was quantified by ELISA, The experiment wasperformed over the siRNA concentration (0, 18, 45, and 90 nM) and overthe NP ratio (0, 10, 15, and 20), which was the ratio of amine in thecationic carrier to phosphate of nucleotide. To investigate weatherintracellular mRNA was reduced selectively, each ionic complex wastreated to cancer cells for 5 hours. 18 hours later, RNA was extracted,followed by PCR to measure the level of intracellular VEGF mRNA.

As a result, the siRNA multi-conjugate of the present invention couldform a stable and even ionic complex with a cationic polymer, comparedwith the conventional siRNA, and demonstrated excellent gene deliveryefficiency and target gene inhibition activity (see FIG. 7).

Experimental Example 4: Investigation of VEGF Inhibition Efficiency

Electrophoresis was performed with the VEGF siRNA multi-conjugateprepared by the method of FIG. 1B, and siRNA was sorted over the size bygel separation method, followed by electrophoresis to confirm thereof.Each siRNA was mixed with Linear PEI to form a complex. 90 nM of thesiRNA conjugate was treated to PC3 cell, and then VEGF gene inhibitioneffect was measured.

As a result, as molecular weight of the siRNA multi-conjugate increased,charge density was increased, suggesting the improvement of genedelivery efficiency using a cationic polymer (see FIG. 8).

Experimental Example 5: Induction of Immune Response by siRNAMulti-Conjugate

To investigate immune response induction capacity of the siRNAmulti-conjugate prepared by the method of FIG. 1B, peripheral bloodmononuclear cells (PBMC) were separated from human blood by using Fisherlymphocyte separation medium. An ionic complex was prepared from theconventional VEGF siRNA or the siRNA multi-conjugate linked by cleavabledisulfide bond or non-cleavable covalent bond by using the cationic genecarrier linear PEI, jet PEI or DOTAP. The siRNA complex was treated toPBMC at the final concentration of 360 nM for 24 hours. INF-alpha levelin the supernatant was measured by ELISA.

As a result, the siRNA multi-conjugate of the present invention did notinduce INF-alpha significantly, compared with the conventional siRNA. Inparticular, the siRNA multi-conjugate prepared by disulfide bonddemonstrated similar INF-alpha induction to the conventional siRNA (seeFIG. 9A).

To confirm whether the prepared siRNA multi-conjugate could induceINF-alpha secretion in mouse, 40 μl of the conventional siRNA or thesiRNA multi-conjugate prepared by cleavable disulfide bond ornon-cleavable covalent bond was mixed with the cationic gene carrierlinear PEI to form an ionic complex, which was injected intravenouslyinto ICR mouse at 7 weeks. After 6 hours of the treatment, blood wastaken from the heart of the mouse, followed by ELISA to quantify bloodsiRNA.

As a result, compared with the conventional siRNA, the siRNAmulti-conjugate of the present invention did not induce INF-alphasecretion significantly in the animal model (see FIG. 9B).

INDUSTRIAL APPLICABILITY

The siRNA multi-conjugate of the present invention can be applied inmedicinal field including gene therapy owing to the improved genedelivery efficiency and thereby further contributes to the advancementof national industry by realizing diverse applications thereof inrelated fields.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

The invention claimed is:
 1. A purified single-stranded homodimeric RNAhaving the structure

wherein each

is a subunit of RNA; wherein each of the subunits is identical; whereinand • and ∘ are distinct functional groups mediating a bond between thesubunits; and wherein the bond is mediated by a bivalent cross-linkingagent comprising maleimide, NHS (N-hydroxysuccinimide), vinylsulfone,iodoacetyl, nitrophenyl azide, isocyanate, pyridyldisulfide, hydrazide,or hydroxyphenyl azide.
 2. The purified single-stranded homodimeric RNAof claim 1, wherein the cross-linking agent has a molecular weight inthe range of 100 Daltons to 10,000 Daltons.
 3. The purifiedsingle-stranded homodimeric RNA of claim 1, wherein the cross-linkingagent comprises DTME (dithiobismaleimidoethane), sulfo-SMCC(sulfosuccinimidyl-4 [N-maleimidomethyl]cyclohexane-1-carboxylate), orBM(PEG)₂ (1,8-bis(maleimido)diethyleneglycol).
 4. The purifiedsingle-stranded homodimeric RNA of claim 1, wherein the subunits arebonded 3′ end to 3′ end, or 5′ end to 5′ end.
 5. The purifiedsingle-stranded homodimeric RNA of claim 1, wherein the subunits arebonded 3′ end to 5′ end.
 6. The purified single-stranded homodimeric RNAof claim 1, wherein subunits are sense or antisense siRNAs.
 7. Thepurified single-stranded homodimeric RNA of claim 1, wherein thesubunits are complementary to c-myc, c-myb, c-fos, c-jun, bcl-2, VEGF,VEGF-B, VEGF-C, VEGF-D, or PIGF mRNA.
 8. The purified single-strandedhomodimeric RNA of claim 1, wherein each of the subunits has 15-50nucleotides or 15-29 nucleotides.
 9. A purified single-strandedhomodimeric RNA having the structure

wherein each

is a subunit of RNA; wherein each of the subunits is identical; whereinand • and ∘ are distinct functional groups mediating a bond between thesubunits; and wherein the bond is a reductant cleavable bond, abio-cleavable bond, or an enzyme cleavable bond.
 10. The purifiedsingle-stranded homodimeric RNA of claim 9, wherein the subunits arebonded 3′ end to 3′ end.
 11. The purified single-stranded homodimericRNA of claim 9, wherein the subunits are bonded 5′ end to 5′ end. 12.The purified single-stranded homodimeric RNA of claim 9, wherein thesubunits are bonded 3′ end to 5′ end.
 13. The purified single-strandedhomodimeric RNA of claim 9, wherein subunits are sense or antisensesiRNAs.
 14. The purified single-stranded homodimeric RNA of claim 9,wherein the subunits are complementary to c-myc, c-myb, c-fos, c-jun,bcl-2, VEGF, VEGF-B, VEGF-C, VEGF-D, or PIGF mRNA.
 15. The purifiedsingle-stranded homodimeric RNA of claim 9, wherein each of the subunitshas 15-50 nucleotides or 15-29 nucleotides.
 16. The purifiedsingle-stranded homodimeric RNA of claim 9, wherein the bond is areductant cleavable bond.
 17. The purified single-stranded homodimericRNA of claim 16, wherein reductant cleavable bond is disulfide.
 18. Thepurified single-stranded homodimeric RNA of claim 17, wherein thesubunits are bonded 3′ end to 3′ end, or 5′ end to 5′ end.
 19. A methodof synthesizing a purified single-stranded homodimeric RNA according toclaim 1, the method comprising covalently bonding

to a bivalent cross-linking agent comprising maleimide, NHS(N-hydroxysuccinimide), vinylsulfone, iodoacetyl, nitrophenyl azide,isocyanate, pyridyldisulfide, hydrazide, or hydroxyphenyl azide, therebysynthesizing a single-stranded homodimeric RNA.
 20. A method ofsynthesizing a purified single-stranded homodimeric RNA according toclaim 9, the method comprising covalently bonding

thereby synthesizing the purified single stranded homodimeric RNA,wherein the bond is a reductant cleavable bond, bio-cleavable bond, orenzyme cleavable bond.